Article in - Hacettepe Journal of Biology

Ö.G.Çelemli / Hacettepe J. Biol. & Chem., 2015, 43 (1), 49–57
Chemical Classification of Propolis Samples Collected from
Different Regions of Turkey in Geographical Region Base
Türkiye’nin Farklı Bölgelerinden Toplanan Propolis Örneklerinin
Coğrafik Bölge Bazında Kimyasal Olarak Sınıflandırılması
Research Article
Ömür Gençay Çelemli
Hacettepe University, Faculty of Science, Department of Biology, Beytepe, Ankara, Turkey
ABSTRAC T
T
his study aims to determine chemical compounds of the propolis samples gathered from different regions
of Turkey in geographical region-base. Gas Chromatography-Mass Spectrometry (GC-MS) analysis, was performed on 21 propolis samples acquired from different geographical regions of Turkey. Samples were collected
from five different geographical regions including Black Sea, Central Anatolia, East Anatolia, Marmarean and
Mediterranean Regions (Three different phytogeographic regions: European-Sibaerian, Irano-Turanian and
Meditteranean phytogeographic regions). So, by this study the chemical compositions of different propolis
samples gathered from three distinct phytogeographical regions will be compared. Accordingly, “1-Nonadecene”, “pinocembrin”, “phenylethyl alcohol”, “2-propen-1-one,1-(2,6-dihydroxy-4-methoxy phenyl)-3-phenyl”
and particularly “ 2-methoxy-4-vinyl phenol”, “ethyl oleate” were determined as mostly found compounds
in collected propolis samples. According to the GC-MS analysis results, five geographical regions are distinguished from each other according to chemical compound groups. “Benzoic acid “and “17-Pentatriacontene”
were found as possible markers of Black Sea Region, “chrysin” for Marmarean region, “2-buten-1-ol,2-methyl”,
“Z-12-pentacosane”, “1-Hexacosane” for Central Anatolia, “Nonadecane”, ”Octadecane”, and “2-Nonadecanone” for East Anatolia Regions. Besides these for the chemical compositions of Mediterranean Region samples,
no clear distinction could be found.
Key Words
Propolis, GC-MS, phytogeographic region, chemical compound, marker compound
ÖZET
B
u çalışma, Türkiye’nin farklı bölgelerinden toplanan çeşitli propolis örneklerinin kimyasal içeriklerini
coğrafik bölge bazında belirlemeyi amaçlamıştır. Türkiye’nin farklı coğrafik bölgelerinden toplanan 21 propolis örneği üzerinden Gaz Kromatografisi-Kütle Spektrometresi (GC-MS) analizi gerçekleştirilmiştir. Örnekler
Karadeniz, İç Anadolu, Doğu Anadolu, Marmara ve Akdeniz Bölgelerini içeren beş farklı coğrafik bölgeden (üç
farklı fitocoğrafik bölge; Avrupa-Sibirya, İran-Turan ve Akdeniz fitocoğrafik bölgeleri) toplanmıştır. Böylelikle
bu çalışmayla, üç farklı fitocoğrafik bölgeden toplanan propolis örneklerinin kimyasal içerikleri kıyasalanmış
olacaktır. Sonuçlara gore, “1-Nonadesin”, “pinosembrin”, “feniletil alkol”, “2-propen-1-on,1-(2,6-dihidroksi4-metoksi fenil)-3-fenil” ve özellikle “ 2-metoksi-4-vinil fenol”, “etil oleat” toplanan propolis örneklerinde en
sık rastlanılan bileşikler olarak saptanmıştır. GC-MS analiz sonuçlarına göre beş coğrafik bölge kimyasal bileşik
grupları bakımından birbirinden ayrılmıştır. “Benzoik asit“ and “17-Pentatriakonten” Karadeniz bölgesi için,
“krisin” Marmara bölgesi için, “2-büten-1-ol,2-metil”, “Z-12-pentakosan”, “1-Hekzakosan” İç Anadolu Bölgesi
için, “ Nonadekan”, ”Oktadekan”, ve “2-Nonadekanon” Doğu Anadolu Bölgesi için olası belirleyici bileşikler
olarak bulunmuştur. Bunların yanında Akdeniz Bölgesi örneklerinde kimyasal içerik bakımından net bir ayrım
bulunamamıştır.
Anahtar Kelimeler
propolis, GC-MS, fitocoğrafik bölge, kimyasal bileşik, belirleyici bileşik
Article History: Received: Aug 22, 2014; Revised: Sep 13, 2014; Accepted: Nov 27, 2014; Available Online: June 20, 2015.
DOI: 10.15671/HJBC.20154311204
Correspondence to: Ö. G. Çelemli, Hacettepe University, Science Faculty, Department of Biology, 06800, Ankara, Turkey.
Tel: +90 312 297 71 61
Fax: +90 312 299 20 28
E-Mail: [email protected]
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Ö.G.Çelemli / Hacettepe J. Biol. & Chem., 2015, 43 (1), 49–57
Figure 1. The collected areas of propolis samples are shown with black circles.
INTRODUCTION
P
ropolis (bee glue) is a sticky dark-coloured
material that honeybees collect from living
plants, mix with wax and use in the construction
and adaptation of their nests. Its resistance
against micro-organisms is an essential
characteristic of propolis and it has been used
by human beings since ancient times for its
pharmaceutical properties [1]
Chemical composition of propolis depends
upon the phytogeographic characteristics of the
site of collection. Although it is of plant origin,
propolis is a bee product. Bees collect it from
different source plants in different ecosystems
by choosing appropriate representatives of the
local flora. Different plant choices of bees as a
source of propolis in different habitats complicate
propolis standardization [2].
For example, while European propolis
contains phenolics: flavonoid agylcones (flavones
and flavonones), phenolic acids and their esters,
the main compound classes found in Brazilian
propolis are prenylated derivatives of p-coumaric
acid and acetophenone [3].
Frequently found as major constituents
in propolis samples are flavonoids, organic
acids, phenols and various kinds of enzymes,
vitamins and minerals. These compounds give
antibacterial, anti-inflammatory, antioxidant,
prooxidant, immuno-enhancement and antitumor
qualities to propolis. For instance, flavonoids, as
antioxidants, prevent oxidative damage of DNA
caused by reactive oxygen species [4].
In Turkey the first research on propolis
was carried out by Sorkun and Bozcuk [5]. They
investigated the effects of propolis on seed
germination. Sorkun et al., also investigated
the chemical composition of propolis samples
taken from Erzurum, Gümüşhane and Trabzon
by another study” [6]. More recently, Kolankaya
et al., [7] studied fat variation and liver injury in
mice that were fed propolis.
Due to the location of Turkey, different
climatic conditions and plant covers can be
seen in this country. Turkey has three phytogeographical and seven geographical regions.
There are 9222 naturally grown species in Turkey
and 3000 of these are endemic [8]. Because of its
rich plant cover and different climatic conditions,
the content of Turkish propolis varies from one
geographical region to another.
Ö.G.Çelemli / Hacettepe J. Biol. & Chem., 2015, 43 (1), 49–57
MATERIALS AND METHODS
Propolis samples
Propolis samples were obtained from the beehives
that were located at different geographical
regions of Turkey (Figure 1).
Extraction and sample preparation
Each frozen propolis sample was ground and
dissolved in ethanol (96%) with a ratio of 1:3 (w/v).
Then the mixture was kept in a tightly closed
bottle in an incubator at 30°C for two weeks.
Then the supernatant was filtered twice through
Whatman (Whatman, Maidstone, England) No.
4 and No. 1 filter papers. The final filtered and
concentrated solution (1:10, w/v) designated
“ethanol extract of propolis” (EEP) and analysed
by gas chromatography and mass spectrometry
(GC-MS) [9].
GC-MS analysis
A GC 6890N instrument from Agilent (Palo
Alto, CA, USA) coupled with a mass spectrometer
(MS5973; Agilent) was used for the analysis of
EEP samples. Experimental conditions of the GCMS system were as follows: a DB 5MS column (30
m x 0.25 mm, 0.25 µm film thickness) was used
and the flow rate of the mobile phase (He) was
set at 0.7 mL/min. In the GC part, temperature
was kept for 1 min at 50°C and then increased to
150°C at 10°C/min intervals followed by 2 min at
150°C. Finally, the temperature was increased to
280°C at 20°C/min intervals and kept at 280°C
for 30 min.
Organic compounds in propolis samples were
identified in Wiley’s NIST Mass Spectral Library,
if the obtained comparison scores were higher
than 95%. Otherwise, fragmentation peaks of the
compounds were evaluated, and the compounds
were identified using the memory background
for the identification of the compounds that
appeared in GC-MS chromatograms. For the
quantification of the compounds in the ethanol
extract, no internal and external standard was
used. Only percentage reports of the compound
in the sample were used. This was the standard
way to quantify the many organic compounds
Figure 2. The PCA analysis of 21 propolis samples in
geographical region base.
in the propolis samples. Contents of individual
compounds in the ethanol extract are given in
percent of the total compounds in the sample.
This was the standard way to quantify most
organic compounds in the propolis samples. In
this case, the relative error could not be higher
than 5% [9].
Statistical analysis
PCA (Principal component analysis) technique
was applied to chemical data for propolis to
evaluate possible similarities and differences
among the propolis samples of five different
geographical regions. In this context, the cases
were the different propolis samples, the variables
were the identified volatile compound groups and
the input value in the matrix was the compound
ratio. The result scores were used for CVA analyse.
RESULTS
According to the results, compounds belong to
alcohols, aldehydes, aliphatic acids and their
esters, carboxylic acids and their esters, cinnamic
acids and their esters, ethers, flavonoids,
hydrocarbons, ketones and terpenes groups were
detected in the collected 21 propolis samples.
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Ö.G.Çelemli / Hacettepe J. Biol. & Chem., 2015, 43 (1), 49–57
Table 1. The propolis samples, collected regions, locations and symbols.
Europaen Siberian
Phytogeographical Region
Irano-Turanian
Phytogeographical Region
Black Sea
Marmarean
Central
Anatolia
East
Anatolia
2-methoxy-4-vinylphenol
5
6
2
3
Phenylethyl alcohol
4
5
2
Benzenemethanol
3
4
Compounds
Mediterranean
Phytogeographical Region
Mediterrranean
Alcohols
2-buten-1-ol,2-methyl
2
3-buten-1-ol,3-methyl
2
3
2-Naphthalenemethanol,1,
2,3,4,4a,5,6,8a-octahydro-.
alpha.,.alpha.,4a,8tetramethyl-,(2R-(2.alpha.,
4a..alpha.,8a.beta.))
2
3
2
5
Aliphatic acids
Ethyl oleate
3
6
Hexadecanoic acid ethyl
ester
4
2
Flavonoids
4H-1-Benzopyran-4-one,5hydroxy-7-methoxy-2-phenyl
3
Chrysin
2-propen-1-one,1(2,6-dihydroxy-4methoxyphenyl)3-phenyl
4
2
4
4
Pinocembrin
3
3
5
3
4
5
3
Hydrocarbons
17-pentatriacontene
3
1-Nonadecene
3
Tricosane
2
4
Z-14-Nanacosane
3
Z-12-Pentacosane
2
1-Hexacosane
2
3
Nonadecane
3
Octadecane
3
2-Nonadecanone
4
Carboxylic acids
Benzoic acid
3
Terpenes
Ketones
Hydrocarbons
Flavonoids
Ethers
Cinnamic
acids and
their esters
Carboxylic
acids and
their
esters
Aliphatic
acids and
their esters
Aldehydes
Alcohols
Comp.
-
0.06
6.21
3.6
-
-
1.51
0.75
0.3
0.51
B1
0.88
0.08
12.11
12.74
0.03
3.29
0.11
3.31
1.12
2.54
B2
-
-
6.62
14.09
-
6.17
8.3
-
0.33
19.21
B3
-
-
5.35
2.72
-
4.73
3.19
0.2
0.38
12.25
B4
1.55
-
6.14
-
6.58
7.78
1.36
0.74
34.6
B5
1.71
9.24
35.77
0.5
-
0.65
7.38
-
6.56
C1
0.44
1.17
4.84
7.17
-
-
1.28
30.73
-
4.54
C2
-
-
5.92
52.52
-
-
-
1.6
-
4.3
C3
-
-
16.82
6.3
-
-
22.5
0.67
E1
0.09
0.97
6.29
47.04
-
-
-
1.27
0.14
12.54
E2
Table 2. The chemical compound groups and ratios (%) determined in propolis samples by GC-MS.
0.12
3.54
5.54
30.71
-
-
0.54
2.89
0,03
5.87
E3
1.52
5.13
24.18
-
-
1.49
22.65
0.45
8.31
E4
0.12
1.82
7.19
0.13
-
0.11
3.29
0.76
4.38
E5
-
2.78
10.42
22.54
-
2.41
2.47
4.10
0.47
29.6
Ma1
-
-
3.73
8.36
-
0.5
7.97
4.31
-
Ma2
0.56
0.3
6.72
-
0.48
9.02
5.15
0.19
Ma3
-
0.51
0.5
10.81
-
26.75
7.3
4.34
0.14
8.39
Ma4
0.31
0.16
6.32
-
0.49
0.34
4.19
0.43
31.11
Ma5
-
-
15.73
4.24
-
-
2.36
15.99
-
13.34
Ma6
-
-
-
-
1.06
1.33
-
Me1
-
0.4
1.39
31.79
0.46
3.47
7.96
0.9
0.14
5.45
Me2
Ö.G.Çelemli / Hacettepe J. Biol. & Chem., 2015, 43 (1), 49–57
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Ö.G.Çelemli / Hacettepe J. Biol. & Chem., 2015, 43 (1), 49–57
As shown in Figure 2, five geographical
regions are distinguished from each other
according to chemical compound groups.
Figure 2 showed the scores scatter plot,
representing the 21 propolis samples.
In general, flavonoids were found in relatively
higher amounts in propolis samples of almost all
the geographical regions. In the case of lower
flavonoid contents, it was observed that these
samples include higher aliphatic acids as well as
hydrocarbons. According to the GC-MS results,
flavonoid group compounds were found in Black
Sea, Central Anatolia, East Anatolia and Marmara
Region samples in high amounts. Contrary to
these samples, in Me1 (Mediterrranean 1) sample
flavonoids couldn’t be observed. While Me1
sample had no flavonoid content, its aliphatic acid
and their esters content were considerably high
(40.55%). Although there was not any flavonoid
content in Me1 sample, a high amount of flavonoid
was observed in the other Mediterranean sample
(Me2) (Table 2). Even though flavonoids were
found in higher amounts in nearly all of the
investigated samples, not all of them are marker
compounds of these samples.
principal compounds of the collected Central
Anatolian samples.”
The identified compounds in more than two of
the five East Anatolian samples were reported in
Table 3. Of these acknowledged compounds, only
ethyl oleate was found in all of the East Anatolian
samples. Besides this, “hexadecanoic acid ethyl
ester, pinocembrin, 2-Nonadecanone were found
nearly in all of the East Anatolian samples (four of
five samples).
According to Table 3 “Ethyl Oleate,
2-methoxy-4-vinylphenol” were found in all of
the six Marmarean samples. These compounds
can be considered as specific to the investigated
Marmarean propolis samples. In addition
to
these
compounds
“benzenemethanol,
phenylethyl alcohol, chrysin, pinocembrin, 4H-1Benzopyran-4-one,5-hydroxy-7-methoxy-2-phenyl,
1-Nonadecene and tricosane were found in more
than three of the six samples.
Only two propolis samples could be
investigated from the Mediterranean Region. Even
though they were located in the same geographical
region, they showed differences from each other
in chemical composition base. For example, while
When the results were compared according to
the content of aliphatic acids and their esters
the geographical region base, it was found that
in Me1 sample is 40.55%, the same content is
“2-methoxy-4-vinyl phenol” could be considered
only
0,9% in Me2 sample. Another variation was
as a principal compound of the investigated Black
observed in flavonoid contents. While Me1 sample
Sea Region samples, since it was present in all
had no flavonoid content, Me2 sample had a
of the five samples collected from this region. In
addition to this, ” Phenyethyl alcohol, 2-propen- rather high flavonoid content (31.79%). Although
1-one,1-(2,6-dihydroxy-4-methoxy
phenyl)-3- these two samples were situated in the same
geographical and phytogeographical region, they
phenyl” were found in four of the five samples
showed many differences from each other. Owing
(Table 3). As these compounds were found
to these results, it can be said that except for
in the samples belong to the other geographical
“Hexadecanoic
acid ethyl ester and 1-Nonadecene”,
regions we couldn’t consider these compounds
there are not many common compounds in the
as possible marker compounds for the Black Sea
Mediterranean samples (Table 3).
Region propolis. and its botanical distinction.
In all the three samples collected from Central
Anatolia Region “2-propen-1-one,1-(2,6-dihydroxy4-methoxyphenyl)-3-phenyl and pinocembrin”
flavonoids were observed. Besides these,
“1-Nonadecene, Z-14-Nonacosane” hydrocarbons
were observed in these three samples, too (Table
3). In the light of these results, it can be said
that these two flavonoids and hydrocarbons are
DISCUSSION
There are many propolis types in the world: Pacific
(macaranga-derived), Mediterranean (containing
mainly diterpenes), South American (Cuban,
Brazillian, Mexican) and red propolis (dalbergiaderived) [2]. Similar to these types, by multiplying
and improving these kind of researches the
Ö.G.Çelemli / Hacettepe J. Biol. & Chem., 2015, 43 (1), 49–57
identification of a type for Turkish propolis can
be made.
If we look at the results in country-base it
can said that firstly, “ 2-methoxy-4-vinyl phenol”,
“ethyl oleate (oleic acid, ethyl ester)” and secondly,
“1-Nonadecene”, “pinocembrin”, “phenylethyl
alcohol”,
“2-propen-1-one,1-(2,6-dihydroxy-4methoxy phenyl)-3-phenyl” compounds can be
considered as possible markers for the collected
Turkish propolis samples, since these compounds
can be found in more than 10 of 21 investigated
samples. From these compounds; Phenylethyl
alcohol and 2-methoxy-4-vinyl-phenol are
alcohols, ethyl oleate is a kind of aliphatic acid and
1-Nonadecene is a hydrocarbon. Of these marker
compounds “pinocembrin and 2-propen-1-one,
1-(2,6-dihydroxy-4-methoxy
phenyl)-3-phenyl
belong to the flavonoid groups. Previous studies
and our researche have showed that Turkish
propolis samples are rich for flavonoids [6,9].
As seen in Table 3, some compounds were
identified as marker compounds for more than
one geographical region. Only a few of these were
found as markers in just one geographical region.
For example benzoic acid and 17-Pentatriacontene
were found as markers of Black Sea Region only. In
Marmarean region “chrysin”, in Central Anatolia
“2-buten-1-ol, 2-methyl”, “Z-12-pentacosane”,
“1-Hexacosane”, in East Anatolia “ Nonadecane”,
”Octadecane”, “2-Nonadecanone” were found
as marker compounds that did not overlap with
other region markers. In the Mediterranean only
“Hexadecanoic acid ethyl ester and 1-Nonadecene
were pinpointed as markers but these two
compounds were also found as markers in the
other regions too. So, no clear distinction could
be found for the Mediterranean Region.
Besides detecting principal compounds we
noticed that Turkish propolis has a quite high
flavonoid content. Similiar to this study Girgin
et al., [10] analysed Zonguldak, Artvin and Bursa
propolis samples too. They also found flavonoids
in different ratios (0.33-19.89%) in these samples
just like us.
Pinocembrin; a kind of flavonoid is the most
abundant compound in the investigated propolis
samples. We found pinocembrin in Çanakkale
(Ma6), Tahtaköprü-Bursa (Ma5), Karacabey-Bursa
(Ma4), Kırklareli (Ma1), Tekirdağ (Ma3), Kayseri
(C2), Ankara (C1), Sivas (C3), Erzincan (E3), Tunceli
(E5), Elazığ (E2), Ardahan (E1) samples. Due to
the pinocembrin contents of these samples, they
can show antimicrobial [11], antioxidant and antiimflammatory activities [12]. In recent years, a
lot of new research were undertaken on this
compound, and some findings indicated that
pinocembrin had some protective effects on
ischemic injury [13]. Also pinocembrin content
possibly gives bacteriostatic, anti-mould and antimycotic effects in vitro, and anti Candida, local
anaesthetic, anti-Helicobacter pylori activities in
external use to Turkish propolis. Similiar to our
results in the previous studies this compound was
found in propolis samples collected from Bursa
[14], Erzincan [15], Erzurum [16] and Elazığ [17].
Another flavonoid “chrysin” was identified
as a possible marker of Marmarean propolis. It
is found in four (Karacabey-Bursa, TahtaköprüBursa, Kırklareli, Tekirdağ) of the six Marmarean
samples, and as we had observed, Velikova et
al., [14] found chrysin in Bursa propolis in their
previous studies. Moreover, tumor cytotoxicity
and anti-Helicobacter pylori activity of this
compound is also known [18].
From
flavonoids
“2-Propen-1-one-1-(2,6dihydroxy-4-methoxyphenyl)
3-phenyl”
was
observed as a possible principal compound for
Black Sea, Central Anatolia and East Anatolia
Regions. It was found in 10 of 21 Turkish propolis
samples. Also in previous studies carried out on
Turkish propolis samples, it is detected in high
amounts [19].
Another frequently found compound, after
flavonoids, was hydrocarbons. Yet, hydrocarbon
content did not give any biological activity to
propolis samples [18] contrary to flavonoids.
The other compound observed quite frequently
was “ethyl oleate”. In this study ethyl oleate was
found in 16 of 21 Turkish propolis samples. This
number is rather high and remarkable. It was
observed in Artvin (B2), Zonguldak (B5), Trabzon
(B4), Çanakkale (Ma6), İstanbul (Ma2), Tahtaköprü-
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Ö.G.Çelemli / Hacettepe J. Biol. & Chem., 2015, 43 (1), 49–57
Bursa (Ma5), Karacabey-Bursa (Ma4), Kırklareli
(Ma1), Tekirdağ (Ma3), Kayseri (C2), Ankara (C1),
Ardahan (E1), Tunceli (E5), Malatya (E4), Erzincan
(E3) and Elazığ (E2) samples. Likewise, in the
previous studies this compound was observed in
samples taken from Ankara, Erzincan [15], Bursa
and Trabzon [6].
Another compound frequently found in the
investigated propolis samples is hexadecanoic
acid ethyl ester. It was found in Ardahan (E1),
Tunceli (E5), Erzincan (E3), Elazığ (E2), Hatay
(Me2) and Antalya (Me1) samples and also this
compound found in Hatay sample previously by
Silici [20].
“Benzoic acid “ a kind of carboxylic acid, was
considered as a basis compound of the Black Sea
Region samples only. Similiarly Girgin et al., [10]
found this compound in a sample of Black Sea
Region too.
A vast number of papers dealing with different
aspects of the biological properties of propolis
have been published during the last decades.
However, a considerable amount of them are of
limited usefulness, despite the claims that they
have on “strong”, ”remarkable” or “significant”
activity. The major reason is the lack of basis
for comparison and scientific evaluation of the
results, since they do not refer to the chemical
nature of the studied propolis samples [2]. It is
deficient for the studies of propolis. So, we aimed
firstly to find the chemical composition and the
possible marker compounds of Turkish propolis
samples. But the samples collected from the
different geographical regions are insufficient to
standardize the propolis samples for geographical
region base.
Although, many studies were done to find
the chemical composition of Turkish propolis,
there is not any detailed research on the marker
compounds of propolis collected from different
geographical regions of Turkey. This study will
be a step towards the geographical distinction
of Turkish propolis. And data will be helpful for
further researches to find marker compounds of
Turkish propolis in geographical region base.
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